Fleshing Out Martian Proteins

Imagine having a modern biology lab on another planet. Then imagine putting that lab on a tiny silicon chip. That portable concept –when applied to detecting the building blocks of life, amino acids — is being investigated for future Mars missions.

Prof. Richard Mathies. Mathies is an UC Berkeley professor of chemistry and developer of the first capillary electrophoresis arrays and new energy transfer fluorescent dye labels – both used in today’s DNA sequencers. Credit: UC Berkeley ,Chem.

Biophysicist Richard Mathies and his colleagues hope to proof-test such a method’s exquisite sensitivity by looking at samples from the closest terrestrial analogs to what might eventually be encountered on Mars later this decade. One such place is one of the driest places on Earth, the Atacama desert in Chile, which Mathies’ team will investigate later this year.

The project will develop an instrument that would probe Mars dust for evidence of life-based amino acids. A NASA Astrobiology Science and Technology for Exploring Planets (ASTEP) grant has been awarded to Mathies, along with Prof. Bada at the Scripps Institution of Oceanography and Dr. Grunthaner at JPL, to develop what they call their Microfabricated Organic Analyzer [MOA] for In Situ Exploration of Mars and Other Solar Bodies. This new instrument [MOA] will then be coupled to a previously developed device called the Mars Organic Detector [MOD], which will acquire and prepare a martian soil sample for biological testing.

The MOD itself is already considered a mature platform. The unit applies heat to a soil sample then purifies and concentrates any organics (called polycyclic aromatic hydrocarbons [PAH], organic amines and amino acids )–the traditional stuff of protein building blocks. "Right now, we are able to detect parts per trillion of amino acids in a gram of soil, which is thousands of times better than Viking," Bada said. The second step then looks for a fluorescent signal based on the travel times of the amino acids down a thin capillary. Different amino acids – there are 20 different kinds used by humans – travel down the tube at different rates, which allows partial identification of those present.

But their design looks not only for this chemical signature of amino acids, since it tests for another critical characteristic of life-based amino acids: Biologically derived amino acids on earth are homochiral and they’re all left handed. Amino acids can be made by physical processes in space – they’re often found in meteorites – but they’re about equally left- and right-handed. If amino acids on Mars have a preference for left-handed over right-handed amino acids, or vice versa, they could only have come from some life form on the planet, Mathies said.

When married together, these remote, robotic laboratories support the suite of next-generation experiments to look for evidence of life on Mars. Bada said, "When I started thinking about tests for chirality and first talked to Rich [Mathies], we had conceptual ideas, but nothing that was actually functioning. He has taken it to the point where we have an honest-to-God portable instrument." With its much greater sensitivity compared to other biological tests that were first tried on the 1977 Viking mission, the new instruments are anticipated for flight aboard NASA’s roving, robotic Mars Science Laboratory mission and/or the European Space Agency’s ExoMars mission, both scheduled for launch in 2009.

Astrobiology Magazine had the opportunity to talk with Professor Richard Mathies about his research group’s interest in the Mars Exploration Project.

Astrobiology Magazine (AM): The project is founded on the the hypothesis that extraterrestrial life would be based on amino acid polymers, and that the evidence for such biology is remotely accessible by heating dirt. Did the 1977 Mars Viking experiments not have the required sensitivity to make a determination of whether a signal was coming from an organic source?

Richard Mathies (RM): The Viking GCMS experiment did not have sufficient sensitivity to detect even high levels of organic molecules like amino acids, and the release experiments were based on the remote possibility of the presence of bacterial cells that were alive and could grow under the available conditions.

The Gas Chromatograph/Mass Spectrometer (GCMS) aboard the Viking Landers looked for organic compounds, but found none. The Biology Instrument aboard the Viking Landers included the Labeled Release (LR) and Gas Exchange (GEX) experiments. The Pyrolytic Release (PR) experiment was based on the presumption that mars was bone dry and thus added no water. The Labeled Release (LR) experiment added only one drop of water, placed at the center of the soil sample so that, as it migrated to the edges, a continuum of wetness would be supplied, declining with distance from the center. The Gas Exchange (GEX) experiment added enough nutrient solution such that the entire sample was wetted. See also November 7th 2003 issue of Science "Mars-Like Soils in the Atacama Desert, Chile, and the Dry Limit of Microbial Life" Credit:NASA.

We feel that the presence of amino acids and measuring homochirality – a prevalence of one type of handedness over another – would be absolute proof of extinct or extant life. That’s why we focused on this type of experiment. If we go to Mars and find amino acids but don’t measure their chirality, we’re going to feel very foolish. Our instrument can do it.

AM: It was concluded in 1977 that Mars’ soil was strongly oxidizing, and that this might complicate interpretation. When a soil sample is heated, can the evolved carbon dioxide just become masked by oxidizing soil conditions or is there another inherent flaw that limits the interpretation of adding nutrients or heat to soil as a biological test?

RM: The pyrolysis conditions cause the conversion of amino acids to amines (especially abundant glycine to methyl amine) but these products are buried under the intense carbon dioxide [CO2] and water [H2O] peaks in the mass spectometer traces making the amines undetectable.

This makes the [Viking’s] GCMS experiment about one thousand times less sensitive for amino acid detection than the apparatus we are developing [1].

AM: The ill-fated European Beagle 2 lander was planning to measure carbon isotopes on Mars as a function of temperature. Are there problems you might foresee with interpreting carbon isotopes alone that testing directly for amino acids clears up?

RM: The determination of the source of isotopic ratio signatures is difficult because it is hard to unambiguously attribute a change in isotopic ratio to a biological source.

When a molecule comes in two mirror-image forms, it is termed chiral. The majority of amino acids are chiral molecules (shown above). Amino acids of biological origin are exclusively homochiral which in turn are left-handed. All proteins on Earth are composed of amino acids of the L type, allowing a chain of them to fold up nicely into a compact protein. When scientists synthesize amino acids from nonchiral precursors, the result is always a "racemic" mixture – equal numbers of right- and left-handed forms. Credit: Bernhard Rupp

First off, the changes are often small. Second, you have to compare the observed ratio to an inorganic standard to identify a biological induced change. On earth this is done with reference to the ratios in carbonates. It is not known what standard to use for this comparison on Mars to establish the nonbiotic control.

Our search for amino acids and their chiral ratios is much more interpretable.

The prototypes our group is developing for the organic tests would first measure microscopic quantities of amino acids using fluorescent labeling and capillary electrophoresis (CE) to determine mass and charge, then check for homochirality.

AM: Can you describe what false positives might arise from the first step of just measuring amino acids alone vs. their biological origin (as left-handed chiral) in the second step?

RM: False positives might arise from space craft residues or propellants. These signals should go down as the rover moves away from the landing area or takes samples farther from the craft or deeper into the soil bed.

It is also possible that we might see amines or ammonia that result from decomposition of organic molecules and amino acids. While this might interfere with amino acid detection this result alone would be very interesting in that it indicates that organic compounds are present on Mars.

AM: There has been recent discussion that sulfur isotopic fractionation might be a clear biological differentiator, particularly given the high sulfate concentrations found at the Opportunity site. Are there disadvantages to that isotope test for remote in situ work compared to building the portable lab your group is designing?

The Murchison Meteorite crashed on September 28, 1969, near Murchison, Australia. The meteorite contains minerals, water, and complex organic molecules such as amino acids.Credit: NASA

RM: Studies of sulfur isotopes have the same problems as carbon isotopes and in addition the changes introduced by biological processes are likely to be smaller because of the larger mass of sulfur.

AM: You mentioned the failure of Viking-style experiments to detect life at Chile’s Atacama desert site. Are there plans to run the electrophoresis tests in situ there as one terrestrial calibration point?

RM: The Viking-type GCMS (Gas Chromatograph/Mass Spectometer) was not run in Atacama but would be unlikely to work well there because of the intense carbon dioxide (CO2) emission from carbonates.

We do plan to run the MOD system coupled to the CE (electrophoresis) analyzer (the whole thing is called MOA) sometime this year in the Atacama.

AM: Are there additional plans to calibrate against any of the meteorites, like Murchison, where amino acids were detected?

RM: Yes, we have run the lab based electrophoresis (CE) system and (Mars Organic Detector) MOD on meteorites including Murchison and we observe what appears to be terrestrial contamination on the surface transitioning to racemic mixtures of amino acids in the interior. Such studies will continue to establish the ground truth.

AM: There is new evidence from Opportunity to indicate the soil may be acidic, or have very low pH. If the soil turns out to be strongly acidic, does that affect either the evaluation of its biological prospects or the electrophoresis tests themselves?

RM: The sublimation in MOD (Mars Organic Detector) which is the first stage of our analysis should eliminate all polar contaminants and salt that would be likely to make a capillary electrophoresis analysis of amino acid composition and chirality difficult.

The Atacama desert is one of the driest places on Earth. Vivid colors belie the arid landscape of northern Chile where the Atacama Desert, one of the world’s driest, meets the foothills of the Andes. Here salt pans and gorges choked with mineral-streaked sediments give way to white-capped volcanoes. This scene was acquired by the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) instrument on NASA’s Terra satellite on October 28, 2001. Scientists are searching for traces of microorganisms in this extreme environment. Credit: USGS

AM: Before running a test, you have to get a sample. How do you presently imagine sample collection being most productive, as either subsurface, surface or another method for site sampling?

RM: The sample collection is not under our control and will be a main subsystem of the lander for all experiments. We hope to have the ability to sample from the surface followed by samples from deeper under the surface crust.

Samples from the surface, which is highly oxidizing, are likely to be low in organics. Samples from deeper under the surface will have higher concentrations of organics. This is the pattern we have seen in the Atacama which is the best available Mars analog site and this experience will be used to guide our sampling methods.

What’s Next

Next on Mathies’ agenda is a field trip to Chile and an investigation as to whether amino acid ‘handedness’ can be detected in situ. The reason Chile’s Atacama Desert is so dry and virtually sterile is because it is blocked from moisture on both sides by the Andes mountains and by coastal mountains. At 3,000 feet, the Atacama is 15 million years old and 50 times more arid than California’s Death Valley.

In February, Grunthaner and UC Berkeley graduate student Alison Skelley traveled to the Atacama desert of Chile to see if the amino acid detector – called the Mars Organic Detector, or MOD – could find amino acids in the driest region of the planet.

The MOD easily succeeded. However, because the second half of the experiment – the "lab-on-a-chip" that tests for amino acid handedness – had not yet been married to the MOD, the researchers brought the samples back to UC Berkeley for that part of the test. Skelley has now successfully finished these field experiments demonstrating the compatibility of the lab-on-a-chip system with the MOD.

"If you can’t detect life in the Yungay region of the Atacama Desert," Mathies said, referring to the desert region in Chile where the crew stayed and conducted some of their tests," you have no business going to Mars".